Genetically Edited Animal

ABSTRACT

The present invention relates to a genetically edited animal, especially to a genetically edited pig in which expression or activity of the RELA protein has been modified. Such pigs have at least partial protection against the African Swine Fever Virus. The invention also provides, a cell nucleus, germ cell, stem cell, gamete, blastocyst, embryo, foetus and/or donor cell of a non-human animal comprising a genetic modification which alters the expression or function of RELA protein, methods for editing the genome of animals and methods for screening the efficacy of a pharmaceutical agent in such an animal.

The present invention relates to a genetically edited non-human animal.In particular the invention relates to a genetically edited mammal, andparticularly to a genetically edited pig. The animal is less affected bypathogen infection; in particular the animal is less likely to developdisease pathology upon virus infection. The genetically edited animalhas altered RELA (p65) expression or activity. The altered RELA activityis preferably caused by a mutation affecting the transactivation domainencoding sequences of RELA. Pigs with altered RELA activity can betolerant of viral infection, in particular infection by African SwineFever Virus (ASFV).

BACKGROUND TO THE INVENTION

Infectious disease adversely affects livestock production and animalwelfare, and often has major impacts upon human health and publicperception of livestock production. For example, the costs of existingendemic diseases are estimated as 17% of turnover of livestockindustries in the developed world and 35-50% in the developing world.Furthermore, epidemics, particularly in the developed countries, incurfurther major costs and profound impacts upon the rural economy and onpublic confidence in livestock production. Biomolecular approaches toenhance the underlying genetic ability of an animal to combat infectiousdisease will enhance animal production regimes. Such technologies, alongwith traditional disease control measures, will enable more effectiveand sustainable disease control.

Central to the immune response a mammal activates up pathogen infectionis the gene transcription pathway utilising the transcription factorNFkappaB, which is an obligate dimer protein. This transcription factorregulates, amongst other signalling pathways, the pathogen inducedcytokine response that is central to the animal's immune response uponinfection. The predominant components of the NFkappaB dimer are the RELA(p65) and NFKB1 proteins. The RELA protein is encoded by a gene whichdisplays polymorphic variation within pig species. In particular Africanand Eurasian pigs display different allelic versions of RELA andfunctional studies indicate that the African RELA variant displays areduced NFkappaB activity that is generally equivalent to the animalcarrying only one copy of the ‘Eurasian’ gene. It is known from mousestudies that animals that carry a single copy of the RELA gene areviable.

Pigs succumb to many pathogens including a typically lethal haemorrhagicfever due to infection by ASFV. African swine fever is a highlyinfectious disease of domestic pigs, with virulent isolates causing arapid fatal haemorrhagic fever. However, in contrast to domestic pigs,the porcine species native to Africa tolerate infection.

ASFV is notifiable to the World Organization for Animal Health (01E),placing it in the highest category of infectious animal pathogens. Itexhibits remarkable potential for transboundary spread, and outbreaks indomestic pig populations have a serious socioeconomic impact worldwide.Furthermore, ASF is considered to be the major limiting factor to pigproduction in Africa. ASFV is a large, double-stranded DNA virus and theonly member of the Asfarviridae family, suggesting that it may carrynovel genes that are not carried by other virus families. The ability ofthe virus to persist in one host while killing another geneticallyrelated host implies that disease severity may be, at least in part,modulated by host genetic variation. Such viruses attempt to evade thehost immune response through the action of virus-encoded immunemodulators. ASFV encodes several such factors, one of which interactswith the NFAT and NFkappaB signalling pathways. Sequencing of componentsof these pathways has indicated a very high degree of homology betweenpig species except in the sequence of RELA. In particular, allelicvariation exists within the gene sequence encoding the transactivationdomains of RELA. Palgrave et al. (Journal of Virology, June 2011, p.6008-6014, Vol. 85, No. 12), which is incorporated herein by reference,describes polymorphisms in the RELA gene which seem to correlate withincreased tolerance to ASFV in warthogs.

Such variation in RELA is notable given the absence of sequencevariation that exists in other components of the NFkappaB and similarpathways between pig species, for example PPIA, NFATC1 regulatory domainand NFKBIA genes.

The present invention provides a genetically edited animal that hasaltered RELA expression or activity. These animals can be generated byan efficient biomolecular approach utilising genome editing technology.Differences in NFkappaB activity that reflect the level of p65 activityare likely to affect how an individual animal responds to pathogenchallenge and to other forms of biological stress or insult. Inparticular, this genetic variation is highly likely to impact on howpigs respond to ASFV and, potentially, other viruses in addition toinfection by other forms of pathogen. Additionally, animals with alteredNFkappaB levels or activity are likely to exhibit marked difference inchronic and autoimmune disease severity.

STATEMENTS OF THE INVENTION

In a first aspect the present invention provides a genetically editednon-human animal comprising a genetic modification which alters theexpression or activity of the RELA protein.

RELA protein is a predominant component of the NFkappaB heterodimerictranscription factor. As such, genetic editing which reduces the levelsor activity of RELA will directly affect NFkappaB dependent cellactivities, in particular transcription from NFkappaB induced genes.NFkappaB is a key effector of animal responses to various stresses,including infection. Genetically edited animals with altered RELAexpression or activity will therefore react differently to theirnon-edited counterparts in response to biological stresses or insults,such as infection, chronic and/or autoimmune diseases.

The cDNA sequence of Sus scrofa RELA is shown in FIG. 1 (SEQ ID NO 1),and the amino acid sequence is shown in FIG. 2 (SEQ ID NO 2). The RELAsequences in other animals are available on GenBank, e.g. cow(NM_001080242.2) (SEQ ID NO.: 38) and chicken (NM_205129.1) (SEQ ID NO.:39).

Preferably the expression or function of the RELA protein in thegenetically edited animal is reduced when compared to a correspondingnon-genetically edited animal, i.e. in which the RELA protein ishomozygous ‘wild-type’ (e.g. in pigs homozygous ‘Eurasian wild type’).Overall activity of RELA in an animal can be reduced by reducing theamount of RELA which is present, by reducing the activity of RELA, or acombination of both. The present invention contemplates all of theseoptions. For example, one could reduce RELA levels by knocking out oneallele of the wild-type allele, while the other is left unaltered; insuch an animal there would be approximately a 50% reduction in RELAlevels, but the activity of the remaining RELA would be unchanged.Alternatively, alterations to one or both of the RELA alleles could beintroduced which reduce, but which do not eliminate RELA activity. Insuch an animal the levels of RELA may be substantially unchanged, butRELA activity is reduced because the RELA protein present is lessactive. The third option is that both the level of RELA and its activitycan be reduced, and this can be achieved through a single edit, orthrough multiple edits to the gene and/or regulatory sequences. If RELAactivity in an animal is completely abolished then the animal istypically non-viable, and therefore that is not a preferred option

In a preferred embodiment of the invention the genetically edited animalis a pig. Preferably the animal is of the species Sus scrofa or Susscrofa domesticus. However, the genetically edited animal can be a cow,sheep, goat or chicken, amongst others.

In general, the discussions below will focus on RELA in pigs. However,it should be noted that RELA is highly conserved across animal speciesand thus equivalent editing events could be applied to the genome ofother domestic animal species.

Preferably all cells of the non-human animal contain the genetic edit.This can be achieved, for example, by modifying the single-cell zygote.A genetic editing event is often referred to as an ‘indel’, and thatterm will be used at various instances below. An ‘indel’ can be aninsertion, a deletion or a substitution.

In a preferred embodiment the modification is a modification to thegenome of the animal, especially a modification of the sequence of theRELA gene. The modification could also be to one or more controlsequences which modulate the expression of the RELA gene. Preferably themodification disrupts or alters the RELA gene sequence such that thereis an overall reduction of RELA activity in the animal's cells.

Alternatively, the modification could be separate from the RELA gene andresult in the expression of a modulator of RELA expression, e.g. byinterfering with normal transcription or translation of the gene. Such amodulator could be a siRNA or antisense polynucleotide which is adaptedto reduce expression of RELA. However, this is generally a lesspreferred embodiment of the present invention.

It is generally preferred that the modification involves an alterationto the coding regions of the RELA gene, i.e. corresponding to the cDNAset out in SEQ ID NO 1. The modification may result in a change of oneor more amino acids relative to the wild type Eurasian RELA sequenceshown in SEQ ID NO 2. The modification could, however, be to non-codingsequences such as introns or splice sites.

In one embodiment of the invention, the genetically edited animalcomprises a modification that substantially or completely knocks out theexpression or activity of the RELA protein. In such an embodiment it ishighly preferred that the modification is mono-allelic, i.e. the animalis heterozygous and retains one functional copy of the wild-type allele(RELA⁻/RELA⁺). Where both alleles are abrogated (i.e. a RELA null,homozygous RELA⁻/RELA⁻), and no functioning RELA is produced, the animalis generally non-viable and typically dies in utero. In one embodimentof the invention one RELA allele is abrogated and the other allele isnot edited, i.e. it is wild type RELA. In such an animal there would beapproximately 50% of normal levels of wild type RELA. Alternatively, oneRELA allele could be abrogated and the other could be modified in amanner which does not completely eliminate RELA expression or activity.An abrogating modification could prevent transcription of the RELAprotein, it could result in production of a non-functional mRNA, or itcould result in translation of an mRNA to form a non-functional protein.There are a wide variety of modifications which could be carried out tosubstantially abrogate expression or function of RELA, and they would bereadily apparent to the person skilled in the art. For example, such amodification could delete at least a portion of the RELA gene or itcould cause a frame-shift in the open reading frame (ORF).

Alternatively, the modification alters, e.g. reduces, the expression oractivity of the edited RELA gene or protein from the edited allele. Sucha modification does not completely abrogate the expression or activityof the edited gene or protein from the allele, but, rather, reduces thelevel of RELA expressed in the cell and/or the ability of RELA to induceNFkappaB dependent induced gene expression.

For example, a modification could be made to disrupt regulatorysequences of the RELA gene, and thus reduce RELA expression from thatallele. Alternatively, the RELA gene could be modified such that theRELA protein expressed from it is less active.

In a preferred embodiment the modification reduces the activity of theRELA protein, e.g. by reducing its ability to be activated by proteinmodification (e.g. phosphorylation) or protein/protein interaction. In aparticularly preferred embodiment of the invention the modificationresults in a change in a transactivation domain of the RELA protein.Suitably, the modification can alter an activation site on the RELAprotein, e.g. by removing or modifying a phosphorylation site.

Where a modification to a RELA allele reduces, but does not abrogateexpression or activity of the RELA gene or protein expressed from thatallele, then it is possible, and indeed may be desirable, that thegenetic modification is bi-allelic.

Editing of the RELA gene sequence can suitably be achieved by any one ormore of:

-   -   Deleting at least a portion of the RELA gene;    -   Inserting a sequence into the RELA gene; and    -   Replacing at least a portion of the RELA gene. Such a        replacement is termed an ‘introgression’ or substitution.

As mentioned above, it can be preferred that the modification is locatedin a region of the RELA gene which encodes the transactivation domain ofRELA. In pigs such domains extend from amino acid 431 to 553 of the wildtype RELA protein sequence (unless otherwise stated, nucleic acid andamino acid numbering is with reference to the wild type Sus scrofa RELAcDNA or protein sequences shown in FIGS. 1 and 2). Transactivationdomain 2 extends from amino acid 431 to 521 and transactivation domain 1extends from amino acid no 522 to the C-terminus of the protein at aminoacid 553. More preferably the modification is located in a regionextending from amino acid 448 to 531 of RELA.

In a preferred embodiment the modification causes a change in the aminoacid located at one or more of the following sites of RELA:

-   -   T448    -   S485    -   S531

Suitably the amino acids at two or more of these sites are altered, andoptionally the amino acids at all three of the sites are altered.

The modifications may suitably result in the following changes in theamino acids of RELA:T448A, S485P, and/or S531P. These alterationscorrespond to polymorphisms which have been observed between domesticpigs and warthogs. These polymorphisms correlate with tolerance to ASFVinfection in warthogs. Warthogs contain several other polymorphism butthey do not affect the expressed amino acid sequence.

In a particularly preferred embodiment the genetically edited animal hasa modification which results in a change in the amino acid at position531 of RELA. Experimental data indicates that, of the three polymorphicsites observed in warhogs versus domestic pigs, the change at positionS531 have the most significant role in modulating RELA activity. Thusmodifications to this site are of principal interest. S531 is aphosphorylation site on RELA, and it is highly likely thatphosphorylation of this site has a role to play in the activation ofRELA, and hence NFkappaB.

Thus, in a particularly preferred embodiment of the present invention,the genetically edited animal comprises a modification which inactivatesor destroys the phosphorylation site at amino acid position S531 inRELA, or a corresponding phosphorylation site in RELA of other species.The modification could alter a single amino acid, e.g. changing theserine to another amino acid which is not amenable to phosphorylation,or it could involve deleting or replacing a larger portion of theprotein or making distal changes to the protein which cause aconformational change which inactivates the phosphorylation site.

The present invention provides a genetically edited pig wherein the RELAgene has been edited such that it comprises a sequence which encodes aRELA protein with a sequence as set out in any one of the following (theamino acids at sites 448, 485 and 531 are shown in bold):

One Amino Acid Change Present

LLQLQFDADEDLGALLGNNTDPTVFTDLASVDNSEFQQLLNQGVSMPPHTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGASGLTNGLLSDGEDFSSIADM(change T448A - SEQ ID NO 3)LLQLQFDTDEDLGALLGNNTDPTVFTDLASVDNSEFQQLLNQGVPMPPHTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGASGLTNGLLSDGEDFSSIADM(change S485P - SEQ ID NO 4)LLQLQFDTDEDLGALLGNNTDPTVFTDLASVDNSEFQQLLNQGVSMPPHTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGASGLTNGLLPDGEDFSSIADM(change S531P - SEQ ID NO 5)

Two Amino Acid Changes Present

LLQLQFDADEDLGALLGNNTDPTVFTDLASVDNSEFQQLLNQGVPMPPHTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGASGLTNGLLSDGEDFSSIADM(changes T448A and S485P - SEQ ID NO 6)LLQLQFDTDEDLGALLGNNTDPTVFTDLASVDNSEFQQLLNQGVSMPPHTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGASGLTNGLLPDGEDFSSIADM(changes T448A and S531P - SEQ ID NO 7)LLQLQFDTDEDLGALLGNNTDPTVFTDLASVDNSEFQQLLNQGVPMPPHTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGASGLTNGLLPDGEDFSSIADM(changes S485P and S531P - SEQ ID NO 8)

Three Amino Acid Changes Present

LLQLQFDADEDLGALLGNNTDPTVFTDLASVDNSEFQQLLNQGVPMPPHTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGASGLTNGLLPDGEDFSSIADM(changes T449A, S485P and S531P - SEQ ID NO 9)

The present invention also provides a domestic pig which has been editedsuch that at least a portion of the autologous RELA sequence has beenreplaced with a sequence which encodes the corresponding warthog(Phacochoerus sp.) RELA protein sequence. The inserted nucleic acidsequence can be identical to the warthog sequence or can be anequivalent artificial sequence with synonymous base changes. Preferablythe portion which is replaced in such an introgression includes thesequences encoding S531, and optionally includes the sequences encodingT449 and/or S485.

For example, in one preferred embodiment a portion of the autologousRELA gene has been removed and the following corresponding sequence hasbeen inserted.

(SEQ ID NO 10) GAACCAGGGTGTAcCcATGCCtCCtCACACAGCcGAGCCCATGCTGATGGAaTAtCCTGAGGCcATAACcCGCTTGGTcACAGGcTCgCAGAGACCtCCcGACCCtGCTCCtACTCCtCTGGGGGCCTCgGGGCTgACCAAtGGTCTCCTCcC cGGGGACGAgGACTTC

Such an introgression causes amino acid changes at sites S485 and S531P.This sequence codes for the warthog RELA protein sequence, but it hasbeen slightly altered to introduce some synonymous nucleic acid changesto the warthog DNA sequence. The synonymous changes were made so thatpreferred ZFN and TALEN pairs, which can be used during theintrogression process, are unlikely to bind. The synonymous changes areshown in lower case, while the changes which cause the S485P and S531Palterations are shown in bold.

In another preferred embodiment a portion of the autologous RELA genehas been removed and the following sequence has been inserted:

(SEQ ID NO 11) GTTTGATgCTGATGAGGACCTGGGGGCCCTGCTCGGCAATAACACTGACCCGACCGTGTTCACGGACCTGGCATCCGTCGACAACTCTGAGTTTCAGCAGCTGCTGAACCAGGGTGTAcCcATGCCtCCtCACACAGCcGAGCCCATGCTGATGGAaTAtCCTGAGGCcATAACcCGCTTGGTcACAGGcTCgCAGAGACCtCCcGACCCtGCTCCtACTCCtCTGGGGGCCTCgGGGCTgACCAAtGGTCTCCT CcCcGGGGACGAgGACTTC

Such an introgression causes amino acid changes at sites T448A, S485 andS531P. The lower case and emboldened bases have the same meaning as inSEQ ID NO 10.

Expression levels of RELA can be determined by directly measuring theamount of protein or by measuring the amount of RELA mRNA in a cell ortissue of the non-human animal. There are a number of well-establishedquantitative assays for measuring protein levels, e.g. ELISA.Quantitative measurement of RELA mRNA levels can be measured usingquantitative real-time reverse transcription polymerase chain reaction(qRT-PCR). Techniques for performing suitable ELISA and qRT-PCRtechniques are well known to the person skilled in the art, and suitableantibodies and PCR primers can be readily obtained or generated. Inpreferred embodiments of the invention where the expression levels ofRELA are reduced, but the remaining RELA is fully functional wild-typeSus scrofa RELA, suitably the amount of RELA protein or RELA mRNA isbetween 30% and 70% of normal levels. Preferably the amount of RELAprotein or RELA mRNA is between 40% and 60% of normal levels, and mostpreferably around 50% of normal levels. ‘Normal levels’ are defined asthe levels measured in a control cell, i.e. the same cell type as thecell being tested, under identical conditions, but wherein the controlcell has not been genetically edited.

Such assays can be performed on tissue (e.g. skin) or cells (e.g.primary fibroblasts isolated from skin biopsy or PBMCs isolated fromblood) isolated from the genetically edited animal. A preferred celltype for assaying is the macrophage.

In terms of RELA activity, because RELA is a predominant component ofthe NFkappaB transcription factor, changes to the activity of RELAfollowing genetic modification can therefore be determined using a testwhich assesses the levels of activity of the NFkappaB pathway in a cell.This can be assessed by measuring the level of proteins or mRNAs whichare induced by NFkappaB, e.g. by qRT-PCR or ELISA. The cells or tissuesto be tested can suitably be subjected to a challenge which stimulatesthe NFkappaB pathway, e.g. stress, lipopolysaccharide (LPS),phorbol-12-myristate-13-acetate, hydrogen peroxide, viral infection,cytokines, irradiation or the like. Suitable tissues and cells caninclude pig tissue (e.g. skin) or cells (e.g. primary fibroblastsisolated from skin biopsy, PBMCs isolated from blood) isolated fromedited pigs, with or without stimulation, e.g. by LPS, TNFalpha, CSF1. Alist of genes targeted by NFkappaB can be found in Pahl H L, “Activatorsand target genes of Rel/NFkappaB transcription factors”, Oncogene 1999November 22; 18(49):6853-66 and in Li X, Stark G R; “NFkappaB-dependentsignalling pathways”, Exp Hematol. 2002 April; 30(4):285-96. The personskilled in the art can select suitable markers from these or other genesknown to be activated by NFkappaB. Exemplary indicator genes which couldbe profiled include TNFalpha, SOD2, CSF1, and HMOX1.

Genetically edited animals according to the present invention preferablydemonstrate one or more of the following phenotypes:

-   -   an altered, especially reduced, NFkappaB signalling capacity;    -   an altered, especially increased, disease resilience or        tolerance;    -   an altered immune response; and    -   an altered stress response.

More particularly, beneficial effects of the genetic modification mayinclude improved tolerance to:

-   -   Virus infection, e.g. ASFV infection in pigs.    -   Pathogen infection, other than viral infection; and    -   General or specific stressors.

In a particularly preferred aspect of the present invention thegenetically edited animal is a pig which has improved tolerance to ASFVinfection.

An animal can be said to be more tolerant to infection when the moralityrate, morbidity rate, the proportion of animals showing significantmorbidity (e.g. weight loss or decreased growth rate), the level ofmorbidity or the duration of morbidity is reduced. In the case of ASFVin domesticated pigs, the morbidity rate approaches 100% in naive herds.The mortality rate depends on the virulence of the isolate, and canrange from 0% to 100%. Highly virulent isolates can cause almost 100%mortality in pigs of all ages. Less virulent isolates are more likely tobe fatal in pigs with a concurrent disease, pregnant animals and younganimals. In sub-acute disease, the mortality rate may be as high as70-80% in young pigs, but less than 20% in older animals. Anystatistically significant reduction (e.g. 95% confidence, or 99%confidence using an appropriate test) in the mortality or morbiditybetween a population of genetically edited pigs and a population ofequivalent non-edited pigs when exposed to ASFV of the same virulencelevel (ideally the same isolate) demonstrates improved tolerance.

According to a second aspect of the invention there is provided a cellnucleus, germ cell, stem cell, gamete, blastocyst, embryo, foetus and/ordonor cell of a non-human animal comprising a genetic modification whichalters the expression or function of RELA protein.

Suitably the cell nucleus, germ cell, stem cell, gamete, blastocyst,embryo, foetus and/or donor cell is derived from a non-human animal asset out above. Alternatively, it can be created de novousing the methodsdescribed herein, or by other methods known to the skilled person, whichallow editing of the genome of an animal cell.

According to a third aspect the invention provides a method of producinga genetically edited non-human animal comprising the steps of:

-   -   Providing a non-human animal cell;    -   Editing the genetic content of the cell to create a modification        which alters the expression or activity of the RELA; and    -   Generating an animal from said cell.

The editing step suitably comprises:

-   -   Introducing a site specific nuclease to the cell, the nuclease        being adapted to bind to a suitable target sequence in the RELA        gene;    -   Incubating said cell under suitable conditions for said nuclease        to act upon the DNA at or near to said target sequence; and    -   Thereby induce recombination, homology-directed repair (HDR) or        non-homologous end joining (NHEJ) at or near the target site.

The non-human animal cell can be a somatic cell, a gamete, a germ cell,a gametocyte, a stem cell (e.g. a totipotent stem cell or pluripotentstem cell) or a zygote.

The method can optionally involve cloning, e.g. somatic cell nucleartransfer (SCNT). In such an embodiment the genetic editing event iscarried out on a somatic cell, after which the edited nucleus istransferred to an enucleated egg cell. Typically a population of somaticcells will be edited and cells in which a desired editing event hasoccurred will be used to provide donor nuclei for SCNT. Processes forSCNT have been well described in the art and would be known to theskilled person.

However, it is an advantage of the present invention that editing can beperformed without the need for cloning.

Preferably the method is performed on a zygote. The term ‘zygote’ can beused in a strict sense to refer to the single cell formed by the fusionof gametes. However, it can also be used more broadly to refer to thecell bundle resulting from the first few divisions of the truezygote—this is more properly known as the morula.

It is preferred that the present method is at least initiated, andpreferably completed, in the zygote at the single cell stage. Thisshould result in all cells of the animal containing the same edit. Itis, however, possible that the zygote may divide while the editingprocess is occurring. Depending on when the cell division occursrelative to the stage of the editing process, it is possible that one ofthe following will occur:

-   -   All cells will contain the same edit because they are derived        from the a single cell which was edited before division        occurred;    -   All cells will contain the same edit because identical editing        events occurred in the daughter cells after division occurred;    -   A mosaic of cells with and without editing events is created        because the cell divided before the editing event occurs and        only one daughter cell was edited; and    -   A mosaic of cells with different edits is created because the        cell divided and differing editing events happened in the        daughter cells.

Editing can also be conducted at after the first cell division, and theresults may be of interest. However, this is generally not preferredwhere the desired result is a non-mosaic animal.

Accordingly, in a preferred embodiment the method comprises the stepsof:

-   -   Providing a zygote of the non-human animal;    -   Introducing a site specific nuclease to the zygote which is        adapted to bind to a suitable target sequence in the RELA gene;    -   Incubating said zygote under suitable conditions for said        nuclease to act upon the DNA at or near to said target sequence;        and    -   Generating an animal from said genetically edited zygote.

It should be noted that the site specific nuclease can be introduced toa cell in any suitable form. For example, the nuclease can be provideddirectly into the cell as a functional protein. Alternatively, thenuclease can be provided into the cell in the form of a precursor ortemplate from which the active nuclease is produced by the cell. In apreferred embodiment an mRNA encoding the nuclease is introduced intothe cell, e.g. by injection. The mRNA is then expressed by the cell toform the functioning protein. Using mRNA in this way allows rapid buttransient expression of the nuclease within the cell, which is ideal forthe purposes of genetic editing.

It should also be noted that the term ‘nuclease’ is intended to coverany biological enzyme which creates a single or double stranded cut of atarget nucleic acid. Accordingly, the term includes nickases andrecombinases, as well as more conventional nucleases which cause singleor double stranded breaks.

The method may comprise inserting a heterologous sequence in the RELAgene at the target site. Such a heterologous DNA sequence can replaceand/or disrupt the endogenous DNA sequence. This can be achieved byintroducing a suitable template DNA molecule to the cell, such as singleor double-stranded DNA molecule, which will be inserted by the cell'sDNA repair mechanisms or an exogenous recombinase. Exemplary DNAsequences for insertion are described above, but many others could ofcourse be used.

The genetically edited zygote can be grown to become an embryo andeventually an adult animal. As discussed above, if the editing eventoccurs in the single-cell zygote then all cells of this animal willtherefore comprise the modified RELA gene as all cells of the animal arederived from a single genetically edited cell. If the editing eventoccurs after one or more cell divisions then the resultant animal willlikely be a mosaic for the editing event, in that it will have somecells derived from the edited cell and some cells derived from uneditedcells.

The method may involve characterising the genetic modification which hasoccurred. Suitable methods to achieve this are set out below.

The method can be performed on a plurality of zygotes and the method mayinvolve selecting zygotes in which the desired genetic modification hasbeen achieved.

Where the modification to the zygote is intended to knock out theexpression or activity of the RELA gene or the RELA protein, the methodmay suitably comprise selecting for zygotes in which the modification ismono-allelic. Given that bi-allelic edited zygotes are typicallynon-viable, the method of selection may simply be selecting for zygoteswhich have been edited, but which do survive to birth.

Preferably the nuclease comprises a pair of transcription activator-likeeffector nucleases (TALENs) or zinc finger nucleases (ZFNs). Suchnucleases are well known in the art and comprise a nuclease moiety fusedto a sequence-specific DNA binding moiety. The nuclease activityrequires a pair of the nuclease moieties to form the active nucleasedimer. Such nucleases are well adapted to site-specific cleaving of DNAmolecules, and techniques to target said nucleases to any desiredsequences are known to the skilled person and described below. TheTALENs or ZFNs can be tailored to target suitable sequences to achievethe desired DNA cut. By inducing a cut in the DNA the cell repairs thecut by NHEJ or HDR. The former is an error prone system and thereforecan be used to introduce edits as a result of errors. The latter can beused to introduce a heterologous sequence into the cell.

Alternatively, the nuclease may comprise a nickase. Nickases are likeTALENs or ZFNs in many ways, but they cause only a single strand break.This can be an advantage in inducing accurate homology-directed repair,which is particularly useful in the present invention to create adesired introgression. Nickases are described in Ramirez et al.‘Engineered zinc finger nickases induce homology-directed repair withreduced mutagenic effects’ Nucleic Acids Research, 2012, 1-9doi:10.1093/nar/gks179.

Another option is that the nuclease comprises a recombinase.Recombinases are a group of enzymes which allow very precisemanipulation and editing of DNA. Although they are not currently asversatile as TALENs, ZFNs and nickases, they have significant potentialto allow very tightly controlled editing events. Recombinationcontrolled by recombinases can be used to accurately paste a sequence ofinterest into the RELA gene.

Another option is that the nuclease comprises an RNA-guidedsite-specific nuclease, such as the CRISPR/Cas system described in Conget al. ‘Multiplex Genome Engineering Using CRISPR/Cas Systems’, Science,15 Feb. 2013: Vol. 339 no. 6121 pp. 819-823. Such systems use an RNAmolecule to target a specific sequence in to be cleaved by the nuclease,in the case of the CRISPR/Cas system ‘spacer’ sequences are used totarget the nuclease. Suitable spacers can be created and used to targetthe Cas nuclease to the desired location in the RELA gene and thus causedouble-stranded breaks in the DNA whereupon NHEJ would result in theintroductions of indels.

Of course, in this rapidly developing field, other techniques forgenetic editing are likely to become available. Such techniques could inmany cases be readily adapted for use in the present invention.

Preferably the nuclease is adapted to target sequences in the region ofthe RELA gene which encodes the transactivation domain of the RELAprotein. The regions of particular interest are discussed in more detailabove.

The site specific nuclease can be adapted to target a sequence proximalto the sequences encoding one or more of amino acids T448, S485, andS531. For example the nuclease can be targeted such that they make asingle or double stranded cut within 100 bases, preferably within 50bases, more preferably within 20, yet more preferably within 10 bases,and most preferably within 5 bases bases of the sequence encoding theseamino acids. Non-homologous end joining and/or homology-directed repaircan then be utilised to edit any one of amino acids T448, S485, andS531, any two of amino acids T448, S485, and S531, or all three aminoacids.

Two or more pairs of TALENs, ZFNs or other such nucleases can be adaptedto excise a region of DNA which encodes any one of amino acids T448,S485, and S531, any two of amino acids T448, S485, and S531, or allthree amino acids.

In preferred embodiments the site specific nucleases are TALENs or ZFNsand are adapted to target the sequences shown in FIGS. 4 and 5, i.e. forTALENs GCCCCCCCACACAGCTG (SEQ ID NO 12) and AGTACCCTGAGGCTAT (SEQ ID NO13), and for ZFNs CTGAGGCTATAACTC (SEQ ID NO 14) and GACAGGGTCCCAGAG(SEQ ID NO 15). However, site specific nucleases adapted to target othersuitable target sequences could of course be used.

The method may suitably comprise delivering a single or double-strandedDNA molecule comprising or consisting of a sequence as set out in SEQ IDNOs10 or 11. As mentioned above, this sequence corresponds to a portionof the warthog RELA gene which includes polymorphisms at sites encodingamino acids T448, S485, and S531.

The method may comprise one or more of the step of testing the abilityof the animal to tolerate challenge with a pathogen, e.g. a virus. Forexample, where the animal is a pig, the method may involve testing theability of the genetically edited animal to survive infection with ahighly virulent ASFV.

According to a fifth aspect of the present invention there is provided amethod of screening the efficacy of a pharmaceutical agent or thevirulence of a pathogen including the steps of:

-   -   Exposing said genetically edited animal according to the present        invention to the agent or pathogen; and    -   Measuring an effect of said agent or pathogen on said animal.

SPECIFIC DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The invention will be further described by way of the followingexamples, which are in no way intended to limit the scope of theinvention. The examples refer to the accompanying drawings, in which:

FIG. 1 shows the Sus scrofa RELA cDNA sequence, GenBank accession numberNM_001114281 (SEQ ID NO 1).

FIG. 2 shows the Sus scrofa RELA amino acids sequence, GenBank accessionnumber NP_001107753 (SEQ ID NO 2).

FIG. 3 shows the Sus scrofa RELA primary amino acid sequence showingallelic variation in transactivation domains and Rel homology domain(see also Palgrave et al, 2011). Domestic pig sequence EMBL depositednumber FN999988. Warthog pig sequence EMBL deposited number FN999989.The full domestic pig (Sus scrofa) sequence is provided and underneath,positions where the warthog sequences differs are indicated, i.e. T448A,S485P and S531P (SEQ ID NO 18).

FIG. 4 shows the Sus scrofa genomic sequence showing the editor bindingsites in bold. Sequence locations marked refer to porcine RELA cDNA,Accession number NM_001114281; fully genomic Sus scrofa sequenceaccession number NC_010444, specifically between 5699452 and 5707267 ofAssembly 10.2 (SEQ ID NOS 19 and 29).

FIG. 5 shows a diagram of the Sus scrofa RELA genomic sequence (SEQ IDNO 37) with TALEN (boxes; TALEN11-1L and TALEN11-1R) and ZFN (boxes ZFN1and ZFN2) binding sites identified; relative positioning of PCR primersalso shown (boxes ss p65NJF1 and ssp65NJR1).

FIG. 6 shows gel electrophoresis identification of editing events byCell assay. Amplified products were digested with Cell and assessed bygel electrophoresis. Minor bands lower down the gel below dominant bandindicate mismatch and editing event. The first 2 gels identifyheterozygous (one allele) editing events; third gel identifieshomozygous (bi-allelic events) through sample mixing assay.

FIG. 7 shows exemplar sequence of RELA edited site (indel). Sequencingtrace and sequence interpretation of indels at porcine RELA in twoindividual pigs (#16 and #26) are shown (SEQ ID NOS 32 to 36, relativeto order shown in Fig).

FIG. 8 shows the location of TALEN, ZFN and PCR primers for sequencingamplicon; porcine RELA sequence 769417-769110 accession numberNW_003609513.1 (SEQ ID NO 37).

FIG. 9 shows an example Cell Assay; lanes A2, A12, B6, B14 and B20 wereidentified as having cleavage products consistent with editing events atthe target site. Direct sequencing of the same PCR products confirmedthis interpretation and identified B12, B16, B17 and B18 as having onlywild-type sequence.

FIG. 10 shows specific indels identified in several piglets (SEQ ID NOS19 to 31).

EXAMPLE 1: EDITOR DESIGN AND CONSTRUCTION

Two types of editor were used: TALEN and ZFN. Both were designed totarget the same region of porcine RELA gene.

TALEN: All TALENs were designed using the TALE-NT software and assembledusing methods described in Cermak et al. (2011)—Nucl. Acids Res. (2011)39 (12): e82. Briefly, intermediary arrays were produced for each TALENpair that were compatible for Golden Gate cloning into pC-+63-TALmodified vector (although other vectors such as pC-+231-TAL,RCIscript-+231-TAL, pC-GoldyTALEN or RCIscript-GoldyTALENetc. could beused). Arrays were joined in the above vectors as follows; 150 ng eachpFUS_A, pFUS_B, pLR-X and the desired backbone were mixed in a 20 μldigestion/ligation reaction including 50 units T4 DNA ligase (NewEngland Biolabs) and 10 units Esp3I (Fermentas) in 1×T4 ligase buffer(New England Biolabs). The reaction was incubated in a thermocycler for10 cycles of 5 min at 37° C. and 10 min at 16° C., then heated to 50° C.for 5 min and then 80° C. for 5 min. Two microliters of each reactionwas transformed into E. coli and plated on LB-carbenicillin plates.Plasmid DNA was purified and mRNA synthesized from SacI linearizedRCIscript vectors using the mMessage Machine T3 Kit (Ambion).

The target sequences for the TALENs used in this work are shown in FIGS.4 and 5. It is of course possible that TALENs targeting other sequenceswithin the RELA gene, or indeed elsewhere in the pig genome, could beused in the present invention. The person skilled in the art couldreadily construct TALENs adapted to target essentially any other desiredsequence using the same techniques described above.

ZFN: ZFNs targeted to porcine RELA were purchased from Sigma who useSangamo algorithm to assist design of modules from the Sangamo archiveto assemble the zinc finger proteins and attach the FokI nuclease domainto create the ZFN. The ZFN was supplied by Sigma as mRNA.

ZFNs targeted at other sequences of interest can also be commerciallysourced.

A useful summary of gene editing using site specific nucleases can befound in the recent review by Moira A McMahon, Meghdad Randar & MatthewPorteus, “Gene editing: not just for translation anymore”, NatureMethods, Vol. 9, No. 1, January 2012.

EXAMPLE 2: TARGET SITE DETAILS

Target sites for the designed TALENs and ZFNs (FIGS. 4 and 5) are shownwith reference to the nucleic acid numbering system of the RELA cDNA (asshown in FIG. 1), GenBank accession number NM_001114281 (SEQ ID NO.: 1);the TALEN target site is located between bases 1458 to 1505; the ZFNtarget site is located between bases 1496 to 1432. The full genomic Susscofa genomic sequence NCBI Reference Sequence accession number isNC_010444 Assembly 10.2.

EXAMPLE 3: PRODUCTION OF GENE EDITED EMBRYOS

To establish the frequency of gene editing in pig embryos an in vitroembryo culture experiment was performed.

Pig ovaries were collected, washed with pre-warmed phosphate bufferedsaline at 38.5° C. and follicles aspirated. Oocytes were washed in TLHEPES PVA before culturing in maturation medium for 44 hours (22 hoursplus hormones and 22 hours minus hormones; 38.5° C., 5% CO₂), followedby gentle pipetting to remove cumulus cells and incubation with preparedsperm for 6 hours (38.5° C. in 5% CO₂). Zygotes were transferred toNCSU-23 HEPES base medium and subjected to a single 2-10 μl cytoplasmicinjection of mRNA at 2, 10 or 25 ng/μl. The 25 ng/μl mRNA sampleconsisted of 20 ng/μl of either a TALEN or ZFN pair mRNA and 5 ng/μlEGFP mRNA. The 10 and 2 ng/μl mRNA samples were dilutions of the 25ng/μl sample. Zygotes were cultured in batches for 66 hours (embryoculture medium; 5% CO2, 38.5° C.), following which they were placedindividually into micro-droplets of medium under oil for visualinspection or harvested for genotyping.

The results of these experiments are shown in Table 1 below.

TABLE 1 Frequency of editing events TALEN injections Embryos No edited %edited/analysed % edited/born No. embryos analysed 120 25 21% — No. pigsanalysed  46 8 — 17% ZFN injections No. pigs analysed  9 1 — 11%

EXAMPLE 4: GENOTYPING OF EDITING EVENTS IN EMBRYOS

Gene editing events in porcine embryos were identified by directsequencing of amplified, isolated DNA and through a gel electrophoresisassay. The latter identified mismatch between the two alleles throughdigestion by the Cell enzyme.

DNA was amplified from harvested embryos using the REPLI-g® mini kit,Qiagen®. The REPLI-g® DNA sample was then used as a template for Highfidelity PCR (AccuPrime™ Taq DNA Polymerase High Fidelity, Invitrogen™)using single-stranded oligonucleotidep65NJF1 (GCAATAACACTGACCCGACCGTG)(SEQ ID NO 16) and single-stranded oligonucleotidep65NJR1(GCAGGTGTCAGCCCTTTAGGAGCT) (SEQ ID NO 17) as primers designed to amplifya 308 base pair region (see FIG. 5) of the wild-type porcine RELA genethat overlapped the TALEN and ZFN cut sites. The PCR product was thensent for sequence analysis to allow identification of editing events.Alternatively, the PCR products were cloned into a plasmid andindividual plasmids sequenced allowing heterozygous and mosaic editingevents to be analysed separately. From 56 of the EGFP positive embryos,16 were confirmed to harbour RELA editing events (often termed anindel). Of these edited embryos, 10 harboured a heterozygous mutation inone allele, while the remaining 6 carried a mutation on both alleles.

The presence of mutations in the RELA gene were additionally identifiedusing a Cell assay (SURVEYOR® mutation detection kit, TRANSGENOMIC®).The high fidelity PCR product was denatured/re-annealed before beingsubjected to SURVEYOR® nuclease activity which cuts at base mismatcheshighlighting insertions, deletions and substitutions. The resultingfragments were subsequently separated by gel electrophoresis foranalysis with size differences identifying edited indel events.

EXAMPLE 5: GENERATION OF GENE EDITED PIGS

Gene edited pigs were produced through injection of the TALEN or ZFNmRNA into the cytoplasm of porcine zygotes.

Embryos were produced from Large-White gilts that were approximately 9months of age and weighed at least 120 kg at time of use.Super-ovulation was achieved by feeding, between day 11 and 15 followingan observed oestrus, 20 mg altrenogest (Regumate, Hoechst Roussel VetLtd) once daily for 4 days and 20 mg altrenogest twice on the 5th day.On the 6th day, 1500 IU of eCG (PMSG, Intervet UK Ltd) was injected at20:00 hrs. Eighty three hours later 750 IU hCG (Chorulon, Intervet UKLtd) was injected.

Donor gilts were inseminated twice 6 hours apart after exhibiting heatgenerated following super-ovulation. Embryos were surgically recoveredfrom mated donors by mid-line laparotomy under general anaesthesia onday 1 following oestrus into NCSU-23 HEPES base medium. Embryos weresubjected to a single 2-5 μl cytoplasmic injection of either ZFN orTALEN pair mRNA at 2 ng/μl. Recipient females were treated identicallyto donor gilts but remained un-mated. Following TALEN or ZFN injection,fertilized embryos were transferred to recipient gilts following amid-line laparotomy under general anaesthesia. During surgery, thereproductive tract was exposed and embryos were transferred into theoviduct of recipients using a 3.5 French gauge tomcat catheter.

EXAMPLE 6: GENOTYPING OF EDITING EVENTS IN PIGS

Gene editing events in born piglets were identified by direct sequencingof amplified, isolated DNA and through gel electrophoresis assay. Thelatter identified mismatch between the two alleles through digestion bythe Cell enzyme.

The DNA was extracted from tissue samples (e.g. ear skin biopsy) usingthe DNeasy Blood and Tissue kit, QIAGEN. A sample of purified DNA wasthen used as a template for High fidelity PCR (AccuPrime™ Taq DNAPolymerase High Fidelity, Invitrogen™) using single-strandedoligonucleotide p65NJF1 (GCAATAACACTGACCCGACCGTG—SEQ ID NO 16) andsingle-stranded oligonucleotide p65NJR1 (GCAGGTGTCAGCCCTTTAGGAGCT—SEQ IDNO 17) as primers designed to amplify a 308 base pair region (FIG. 5) ofthe wild-type porcine RELA gene that overlapped the TALEN and ZFN cutsites. The PCR product was then sent for sequence analysis to allowidentification of editing events. Alternatively, the PCR products werecloned into a plasmid and individual clones sequenced allowingheterozygous and mosaic editing events to be analysed separately.

The presence of mutations in the RELA gene were additionally identifiedusing a Cell assay (SURVEYOR® mutation detection kit, TRANSGENOMIC®).The high fidelity PCR product was denatured/re-annealed before beingsubjected to SURVEYOR® nuclease activity which cuts at base mismatcheshighlighting insertions, deletions and substitutions. The resultingfragments were subsequently separated by gel electrophoresis foranalysis with size differences identifying edited indel events.

From 46 born piglets analysed following TALEN pair zygotic injection, 8were identified as being genome edited (Table 1). See FIG. 7 forexemplars of direct sequence analysis from 2 of the TALEN genome editedpiglets. From 9 born piglets analysed following ZFN pair zygoticinjection 1 was identified as being genome edited (Table 1).

The following examples relate to methodology to create pigs withsequences inserted into the RELA gene and to analyse the phenotype ofgenetically edited pigs.

EXAMPLE 7: PRODUCTION OF GENE INTROGRESSION EMBRYOS USING EDITINGTECHNOLOGY

To establish the frequency of gene editing in pig embryos in vitroembryo culture experiment is performed.

Pig ovaries are collected, washed with pre-warmed phosphate bufferedsaline at 38° C. and follicles aspirated. Oocytes are washed in TL HEPESPVA before culturing in maturation medium for 44 hours (22 hours plushormones and 22 hours minus hormones; 39° C., 5% CO₂), followed bygentle pipetting to remove cumulus cells and IVF for 6 hours (38.5° C.in 5% CO₂). Zygotes are transferred to NCSU-23 HEPES base medium andsubjected to a single 2-5 μl cytoplasmic or pronuclear injection ofeither ZFN or TALEN pair mRNA between 2 to 10 ng/μl mixed with from 1 to10 ng/μl (optimum concentrations can be determined by experimenter)single-stranded or double-stranded DNA fragment or plasmid. Zygotes arecultured in batches for 66 hours (embryo culture medium; 5% CO₂, 38.5°C.), following which they are placed individually into micro-droplets ofmedium under oil for visual inspection or harvested for genotyping.

EXAMPLE 8: GENOTYPING OF GENE INTROGRESSION EVENTS IN EMBRYOS

Gene introgression events in porcine embryos are identified by directsequencing of amplified, isolated DNA.

DNA is amplified from harvested embryos using the REPLI-g® mini kit,Qiagen®. The REPLI-g® DNA sample is then used as a template for Highfidelity PCR (AccuPrime™ Taq DNA Polymerase High Fidelity, Invitrogen™)using single-stranded primers designed to amplify replicon containingthe target region. The PCR product is then sent for sequence analysis toallow identification of editing events. Alternatively, the PCR productsare cloned into a plasmid and individual plasmids sequenced allowingheterozygous and mosaic editing events to be analysed separately.

EXAMPLE 9: PRODUCTION OF GENE INTROGRESSION PIGS USING EDITINGTECHNOLOGY

Gene introgression edited pigs are produced through injection of theTALEN or ZFN mRNA in combination with a single-stranded DNA oligo ordouble-stranded DNA fragment into the cytoplasm or nucleus of porcinezygotes.

Embryos are produced from Large-White gilts that are approximately 9months of age and which weigh at least 120 kg at time of use.Super-ovulation is achieved by feeding, between day 11 and 15 followingan observed oestrus, 20 mg altrenogest (Regumate, Hoechst Roussel VetLtd) once daily for 4 days and 20 mg altrenogest twice on the 5th day.On the 6th day, 1500 IU of eCG (PMSG, Intervet UK Ltd) is injected at20:00 hrs. Eighty three hours later 750 IU hCG (Chorulon, Intervet UKLtd) is injected.

Donor gilts are inseminated twice 6 hours apart after exhibiting heatgenerated following super-ovulation. Embryos are surgically recoveredfrom mated donors by mid-line laparotomy under general anaesthesia onday 1 following oestrus into NCSU-23 HEPES base medium. Embryos aresubjected to a single 2-5 μl cytoplasmic or pronuclear injection ofeither ZFN or TALEN pair mRNA at 2 to 10 ng/μl mixed from 1 to 10 ng/μl(optimum concentrations can be determined by experimenter)single-stranded or double-stranded DNA fragment or plasmid.

Suitable sequences are:

(SEQ ID NO 10) GAACCAGGGTGTAcCcATGCCtCCtCACACAGCcGAGCCCATGCTGATGGAaTAtCCTGAGGCcATAACcCGCTTGGTcACAGGcTCgCAGAGACCtCCcGACCCtGCTCCtACTCCtCTGGGGGCCTCgGGGCTgACCAAtGGTCTCCTCcC cGGGGACGAgGACTTC(SEQ ID NO 11) GTTTGATgCTGATGAGGACCTGGGGGCCCTGCTCGGCAATAACACTGACCCGACCGTGTTCACGGACCTGGCATCCGTCGACAACTCTGAGTTTCAGCAGCTGCTGAACCAGGGTGTAcCcATGCCtCCtCACACAGCcGAGCCCATGCTGATGGAaTAtCCTGAGGCcATAACcCGCTTGGTcACAGGcTCgCAGAGACCtCCcGACCCtGCTCCtACTCCtCTGGGGGCCTCgGGGCTgACCAAtGGTCTCCT CcCcGGGGACGAgGACTTC

Recipient females are treated identically to donor gilts but remainun-mated. Following TALEN or ZFN plus DNA injection, fertilized embryosare transferred to recipient gilts following a mid-line laparotomy undergeneral anaesthesia. During surgery, the reproductive tract is exposedand embryos are transferred into the oviduct of recipients using a 3.5French gauge tomcat catheter.

EXAMPLE 10: GENOTYPING FOR GENE INTROGRESSION IN EDITED PIGS

Gene introgression events in born piglets are identified by directsequencing of amplified, isolated DNA.

The DNA is extracted from tissue samples (e.g. ear skin biopsy) usingthe DNeasy Blood and Tissue kit, QIAGEN. A sample of purified DNA isthen used as a template for High fidelity PCR (AccuPrime™ Taq DNAPolymerase High Fidelity, Invitrogen™) using single-strandedoligonucleotides p65NJF1 (GCAATAACACTGACCCGACCGTG—SEQ ID NO 16) andsingle-stranded oligonucleotide p65NJR1 (GCAGGTGTCAGCCCTTTAGGAGCT—SEQ IDNO 17) designed to amplify a 308 base pair region (FIG. 5) of thewild-type porcine RELA gene that overlapped the TALEN and ZFN cut sites.The PCR product was then sent for sequence analysis to allowidentification of editing events. Alternatively, the PCR products werecloned into a plasmid and individual clones sequenced allowingheterozygous and mosaic editing events to be analysed separately.

EXAMPLE 11: EVALUATION OF ALTERED RELA LEVELS IN PIG TISSUE AND IN CELLSISOLATED FROM GENE EDITED PIGS

To assess the effect of editing of the RELA locus on RELA levels one canutilise qRT-PCR and RELA protein levels by Western blot and ELISA in pigtissue (e.g. skin) or cells (e.g. primary fibroblasts isolated from skinbiopsy, PBMCs isolated from blood) isolated from edited pigs. Suitableprimers and probes for qRT-PCR can readily be determined by the personskilled in the art.

EXAMPLE 12: EVALUATION OF ALTERED NFKAPPAB SIGNALLING IN PIG TISSUE ANDIN CELLS ISOLATED FROM GENE EDITED PIGS

RELA is a predominant component of the NFkappaB transcription factor. Toassess the effect of editing of the RELA locus on NFkappaB signallingone can perform qRT-PCR for genes known to be activated by NFkappaB onpig tissue (e.g. skin) or cells (e.g. primary fibroblasts isolated fromskin biopsy, PBMCs isolated from blood) isolated from edited pigs withor without stimulation, e.g. by LPS, TNFalpha, CSF1.

EXAMPLE 13: EVALUATION OF ALTERED CELLULAR RESPONSE TO VIRUS CHALLENGEIN CELLS ISOLATED FROM GENE EDITED PIGS

To assess the effect of editing of the RELA locus on the cellularresponse to virus challenge PBMCs from blood are isolated. CulturedPBMCs, either with or without CSF1, are exposed to virus challenge (e.g.influenza virus, PRRSV). The signalling response to virus challenge isassessed by expression profiling.

CONCLUSION

There have been described a number of methodologies to modify (edit) thegenome of pigs. These methodologies can readily be adapted to modify thegenetics of other animals, such as cows, sheep, goats and chickens.

In pigs the modification of RELA can provide increased tolerance againstASFV. This provides a novel mechanism by which tolerance to thisextremely significant pathogen can be created. The commercial andecological importance of this is great. Domestic pig production inAfrica is severely restricted because of the effects of ASFV. Outside ofAfrica, ASFV is endemic in feral pigs in Sardinia, Italy. In 2007 it wasintroduced into the Caucasus, and has apparently become endemic amongwild boars in the region. There have been outbreaks of the virus indomesticated swine in the Republic of Georgia, Russia, Armenia,Azerbaijan and other countries in the region. There is thus a great needto protect against the spread of this disease and also to mitigate therisk should controlling prove impossible. The present invention has thepotential to significantly mitigate the problems should ASFV infectionbecome more widespread and raises the possibility of increasing pigproduction in Africa.

ADDITIONAL EXEMPLIFICATION

Additional work was conducted to further demonstrate the power of theabove mentioned gene editing techniques. In this work it is demonstratedthat both TALEN and ZFN technology can be efficiently applied toengineer pig zygotes that result in gene edited live births, both mono-and bi-allelic (Table 2), significantly broadening the use of editortechnology in livestock.

We performed cytoplasmic editor injection into porcine zygotes, giventhe difficulty associated with visualization of the pronucleus inporcine zygotes. Oocytes were harvested from slaughterhouse material forin vitro studies and superovulated artificially inseminated sows forembryos destined for transfer into recipients. Initially 208 zygoteswere subjected to a single cytoplasmic injection of 10 μl of a solutioncomposed 20 ng/μl RELA TALEN mRNA with 5 ng/μl EGFP mRNA (to enablevisual identification and isolation of embryos that functionallytranslated the injected mRNA). After approximately 3 days of in vitrodevelopment, GFP fluorescence was detected in 36% of embryos. Thus mRNAinjected into the cytoplasm of pig zygotes translates to functionalprotein in the embryo. GFP positive embryos were screened for editingevents by Cell surveyor assay (FIG. 9) and sequencing of PCR amplifiedfragments (shown below and in FIG. 10). We detected 16 editing events in46 GFP-positive embryos analysed (35%). In a second experiment we tested34 embryos injected with 2 μl of 20 ng/μl RELA TALEN mRNA but withoutselection for GFP activity, and detected 2 editing events (6%). In twofurther experiments where 2 μl of 10 ng/μl or 2 μg/μl RELA TALEN mRNAwas injected, 0% and 18% editing frequency, respectively, was observed.Thus, in total we identified 21 editing events in porcine embryos invitro (21% of tested embryos), and a high frequency of these editingevents were biallelic in nature (29% of editing events). Calculatingthis as a frequency of tested embryos, we achieved a biallelic editingfrequency of 6%.

Since both the highest and lowest tested concentrations of RELA TALENmRNA produced edited embryos in vitro we elected to transfer zygotesinjected with each tested TALEN amount into recipient sows and allowpregnancies to develop to term. Pregnancy rates for the higherconcentrations of RELA TALEN mRNA were poor; 1 out of 2 transfers and 0out of 2 transfer for embryos injected with 10 ng/μl or 20 ng/μl,respectively. This poor pregnancy rate reflect the visual observationthat 2 ng/μl RELA TALEN mRNA injected embryos developed better in vitrothan those injected with higher concentrations of TALEN mRNA. The onepregnancy from 10 ng/μl delivered 7 piglets, none of which harbouredRELA editing events by direct sequencing of PCR amplified products. Wedid not pursue transfers of embryos with these higher TALEN mRNAconcentrations any further.

In contrast, transfer of embryos injected with 2 ng/μl RELA TALEN RNAresulted in 5 pregnancies from 7 recipients. One subsequently aborted at15 weeks of pregnancy just prior to parturition; analysis of the 9foetuses carried revealed 3 to have editing events. In total from theremaining 4 farrowings, 39 piglets were produced of which 8 carriedediting events (21%). Of the 8 editing events, 2 animals were stillbornand a further 1 died neonatally due to being crushed by the mother; 5are still alive.

In parallel we tested a ZFN with a target location of 1496 to 1532 bprelative to the translational start site in porcine RELA cDNA sequence(NM_001114281) (SEQ ID NO.: 1). Again the one transfer of embryosinjected with RELA ZFN mRNA at 10 ng/μl failed to generate a pregnancywhile the two transfers of embryos injected with RELA ZFN mRNA at 2ng/μl both became pregnant resulting in the birth of 9 piglets. Of these9 piglets, one carried an editing event at the ZFN target site (11%);although low numbers, this is a comparable frequency in comparison toour observed TALEN editing efficiency.

Direct sequencing of PCR products revealed a variety of editing eventsin piglets derived from TALEN and ZFN injected embryos. Analysis of earbiopsy isolated genomic DNA identified both deletions and insertions atthe target sites. Sequence data from 2 animals constituted multipleoverlapping traces indicating two or more editing events; this wassubsequently confirmed by sequencing of multiple cloned PCR products.Presuming that in these cases of multiple editing the frequency ofevents detected in ear biopsy reflects frequency in the early embryo,designer nuclease editing can remain active beyond the 2-cell stage(i.e. some events display low representation in the PCR pool and aretherefore only present in a subset of cells). In total, 5 biallelicevents where identified from 9 edited piglets (56%; 9% of piglets born);4 from TALEN and 1 from ZFN mRNA injections. Of these bialleleic events2 were homozygous with 3 displaying different indels on each allele.While both piglets carrying homozygous biallelic event survivedfarrowing (milk in stomach post mortem), they both died within the first24 hours of life: the ZFN-derived piglet with an 18 bp biallelichomozygous deletion was bitten by its mother while the TALEN-derivedpiglet with a 6 bp biallelic homozygous deletion was crushed by itsmother.

In summary, we observed an overall editing frequency of 2% oftransferred embryos or 16% of piglets born. These figures comparefavourably with that reported for zygote injection of ZFNs in rats where2% of transferred embryos and 12% of founder animals harboured editingevents (Guerts, A. M. et al. Science 325, 433 (2009)). Our editingfrequencies also compare favourably with the production of monoallelic(0.1% of transferred embryos) and biallelic (1% of transferred embryos;the greater frequency over monoallelic due to incorporation of a FACSenrichment stage prior to somatic cell nuclear transfer (Hauschild, J.et al. Proc. Natl. Acad. Sci. USA 108, 12013-12017 (2011)) pigs usingsomatic cell nuclear transfer methodology.

In conclusion, we demonstrate that editor technology, both TALEN andZFN, can be successfully applied to pig zygotes to produce live geneedited pigs and contrary to predictions the delivery of editors by thedirect injection into the zygote is both efficient and able to generatebiallelic mutations. This novel achievement paves the way for precisegenome engineering of livestock independent of somatic cell nucleartransfer (cloning) technology.

Materials and Methods for Additional Exemplification

Editor Design and Construction

Two types of editor were used: TALEN and ZFN. Both were designed totarget the same region of porcine RELA gene.

TALEN: TALENs were designed using the TALE-NT software and assembledusing methods described previously (Carlson, D. F. et al. Proc. Natl.Acad. Sci. USA 109, 17382-17387 (2012)). Briefly, intermediary arrayswere produced for the porcine RELA TALEN pair for Golden Gate cloning asfollows; 150 ng each pFUS_A, pFUS_B, pLR-X and pC-+63-TAL modifiedvector were incubated for 10 cycles of 5 min at 37° C. and 10 min at 16°C., then heated to 50° C. for 5 min and then 80° C. for 5 min in thepresence of 50 units T4 DNA ligase (New England Biolabs), 10 units Esp3l(Fermentas), lx T4 ligase buffer (New England Biolabs). Plasmid DNA waspurified from ligated vector transformed E. coli and mRNA synthesizedfrom SacI linearized RCIscript vectors using the mMessage Machine T3 Kit(Ambion).

ZFN: ZFNs targeted to porcine RELA were purchased from Sigma. The ZFNdisplayed 84.7% cutting in MEL1 assay (Sigma data sheet) and wassupplied as mRNA.

Zygote Injections

To establish the frequency of gene editing in pig embryos an in vitroembryo culture experiment was performed. Pig ovaries were collected,washed with pre-warmed phosphate buffered saline at 38° C. and folliclesaspirated. Oocytes were washed in TL HEPES PVA before culturing inmaturation medium for 44 hours (22 hours plus hormones and 22 hoursminus hormones; 39° C., 5% CO₂), followed by gentle pipetting to removecumulus cells and IVF for 6 hours (38.5° C. in 5% CO₂). Zygotes weretransferred to NCSU-23 HEPES base medium and subjected to a single 2-10μl cytoplasmic injection of mRNA at 2, 10 or 20 ng/μl+1-5 ng/μl EGFPmRNA. The 10 and 2 ng/μl mRNA samples were dilutions of the 20 ng/μlsample. Zygotes were cultured in batches for 66 hours (embryo culturemedium; 5% CO2, 38.5° C.), following which they were placed individuallyinto micro-droplets of medium under oil for visual inspection orharvested for genotyping either as a total group or after manualselection of GFP fluorescing embryos.

Embryo Transfers

Embryos were produced from Large-White gilts that were approximately 9months of age and weighed at least 120 kg at time of use.Super-ovulation was achieved by feeding, between day 11 and 15 followingan observed oestrus, 20 mg altrenogest (Regumate, Hoechst Roussel VetLtd) once daily for 4 days and 20 mg altrenogest twice on the 5th day.On the 6th day, 1500 IU of eCG (PMSG, Intervet UK Ltd) was injected at20:00 hrs. Eighty three hours later 750 IU hCG (Chorulon, Intervet UKLtd) was injected.

Donor gilts were inseminated twice 6 hours apart after exhibiting heatgenerated following super-ovulation. Embryos were surgically recoveredfrom mated donors by mid-line laparotomy under general anaesthesia onday 1 following oestrus into NCSU-23 HEPES base medium. Embryos weresubjected to a single 2p1 cytoplasmic injection of either ZFN or TALENpair mRNA at 10 ng/μl or 2 ng/μl.

Recipient females were treated identically to donor gilts but remainedun-mated. Following TALEN or ZFN injection, fertilized embryos weretransferred to recipient gilts following a mid-line laparotomy undergeneral anaesthesia. During surgery, the reproductive tract was exposedand embryos were transferred into the oviduct of recipients using a 3.5French gauge tomcat catheter. Litter sizes ranged from 3-17 piglets.

Genotyping

Gene editing events in porcine embryos were identified by directsequencing of amplified, isolated DNA and through a gel electrophoresisassay. The latter identified mismatch between the two alleles throughdigestion by the Cell enzyme.

Sequencing: DNA was amplified from harvested embryos using the REPLI-g®mini kit, Qiagen®. The REPLI-g® DNA sample was then used as a templatefor High fidelity PCR (AccuPrime™ Taq DNA Polymerase High Fidelity,Invitrogen™) using p65NJF1 5′-GCAATAACACTGACCCGACCGTG-3′ (SEQ ID NO 16)and p65NJR1 5′-GCAGGTGTCAGCCCTTTAGGAGCT-3′ (SEQ ID NO 17) as primersdesigned to amplify a 308 base pair region of the wild-type porcine RELAgene that overlapped the TALEN and ZFN cut sites. The PCR product waspurified then sent for sequence analysis to allow identification ofediting events. Alternatively, the PCR products were cloned into aplasmid and individual plasmids sequenced allowing heterozygous andmosaic editing events to be analysed separately.

The following sequences were determined (TALEN/ZFN target sites areshown in bold, insertions are double underlined, and deletions are shownwith symbol 1:

TALEN binding sites (SEQ ID NO 19)GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCT GAGGCTATAACTC WTPiglet 8770-I (SEQ ID NO 19)GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCT GAGGCTATAACTC WT(SEQ ID NO 20) GGTGTATCCATGCCCCCCCACACAGCTGAGCCCA~GCTGATGGAGTACCCTGAGGCTATAACTC Δ1 Piglet 8770-J (SEQ ID NO 19)GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCT GAGGCTATAACTC WT(SEQ ID NO 21) GGTGTATCCATGCCCCCCCACACAGCTGAGCCCA T TGCTGATGGAGTACCCTGAGGCTATAACT +1 Piglet 8770-26 (SEQ ID NO 19)GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCT GAGGCTATAACTC WT(SEQ ID NO 20) GGTGTATCCATGCCCCCCCACACAGCTGAGCCCA~GCTGATGGAGTACCCTGAGGCTATAACTC Δ1 Piglet 8130-sat on (SEQ ID NO 22)GGTGTATCCATGCCCCCCCACACAGCTGA~~~~~~GCTGATGGAGTACCCT GAGGCTATAACTC Δ6Piglet 8130-16 (SEQ ID NO 19)GGTGTATCCATGCCCCCCCACACAGCTGAGCCCATGCTGATGGAGTACCCT GAGGCTATAACTC WT(SEQ ID NO 23) GGTGTATCCATGCCCCCCCACACAGCTGAGCCCTCCATCAGCTGATGGA GTACCCTGAGGCTAT +5 (SEQ ID NO 24)GGTGTATCCATGCCCCCCCACACAGCTGAG~~~~~~~~~~~~~~~~~CCCT GAGGCTATAACTC Δ17Inserted sequence (double underlined) is duplica-tion/inversion of underlined sequence Piglet 8784-30 (SEQ ID NO 25)GGTGTATCCATGCCCCCCCACACAGCTGAGC~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~ Δ111(SEQ ID NO 26) GGTGTATCCATGCCCCCCCACACAGCTGAGCC~~~~CTGATGGAGTACCCTGAGGCTATAACTC Δ4 Piglet 8784-33 (SEQ ID NO 27)GGTGTATCCATGCCCCCCCACACAGCTGAGC~~~~~CTGATGGAGTACCCT GAGGCTATAACTC Δ5(SEQ ID NO 22) GGTGTATCCATGCCCCCCCACACAGCTGAG~~~~~~CTGATGGAGTACCCTGAGGCTATAACTC Δ6 (SEQ ID NO 28)GGTGTATCCATGCCCCCCCACACAGCTGAGCC~~~~CTGATGGAGTACCCT GAGGCTATAACTC Δ4Piglet 8784-34 (SEQ ID NO 20)GGTGTATCCATGCCCCCCCACACAGCTGAGCCCA~GCTGATGGAGTACCCT GAGGCTATAACTC Δ1(SEQ ID NO 29) GGTGTATCCATGCCCCCCCACACAGCTGAGCCC+ TGAGTACCCTGA GGAGTACCCTGA GGCTATA Δ8 + 12Double underlined portion of inserted sequence isinversion of underlined sequence in WT. ZEN binding site (SEQ ID NO 30)TGCTGATGGAGTACCCTGAGGCTATAACTCGCTTGGTGACAGGG TCCCAGA GACCCCCTGACC WTPiglet 8142-C (SEQ ID NO 31) TGCTGATGGAGTACCCTGAGGCTATAACTC~~~~~~~~~TCTGGGA ~~~~~ ~~~~CCCTGACC Δ25 + 7Inserted sequence (double underlined) is inversionof underlined sequence in WT.

Cel1 assay: The presence of mutations in the RELA gene were additionallyidentified using a Cell assay (SURVEYOR® mutation detection kit,TRANSGENOMIC®). The high fidelity PCR product was denatured/re-annealedbefore being subjected to SURVEYOR® nuclease activity which cuts at basemismatches highlighting insertions, deletions and substitutions. Theresulting fragments were subsequently separated by gel electrophoresisfor analysis with size differences identifying edited events.

Comparison of In Vitro Embryo and Piglet Editing Frequency

For RELA TALEN mRNA injected zygotes we transferred 393 embryos into 11recipient sows, resulting in 6 pregnancies and 5 farrowings with littersizes ranging from 3 to 17. One female aborted within last two weeks ofpregnancy. Of the 46 piglets born, 5 were stillborn while of the liveborn 13 were savaged, sat on by the sow or culled on veterinary advice.Post mortem investigation failed to detect any common pathologyassociated with the dead piglets.

In comparison to previous studies describing the combination of editortechnology and somatic cell nuclear transfer, zygote injectionrepresents an efficient technology. In our 32 donor/recipient animalswere used to produce 9 edited piglets. In contrast the generation ofgenome edited pigs by somatic cell nuclear transfer used, for example,approximately 75 donor/recipients to produce 2 edited animals (Yang, D.et al. Cell Res. 21, 979-982 (2011)). while approximately 84donor/recipient animals to produce 11 edited piglets in a report ofbiallelic genome editing (Hauschild, J. et al. Proc. Natl. Acad. Sci.USA 108, 12013-12017 (2011)); assuming 20 oocytes per donor animal.

Table 2 summarises the TALEN-mediated editing events in embryos andpiglets.

TABLE 2 Numbers for TALEN edited indels in porcine embryos in vitro andpiglets. Embryos in vitro GFP PCR Injected fluorescence amplifiedEdited* Biallelic TALEN zygotes (visual) (tested) (% of tested) (% oftested) 20 ng/μl 208 75 46 16 (35%) 5 (11%) 20 ng/μl 68 ND 34 2 (6%) 1(3%) 10 ng/μl 38 ND 3 0 (0%) 0 (0%) 2 ng/μl 53 ND 17 3 (18%) 1 (6%)total 367 NA 100 21 (21%) 6 (6%) Piglets Transferred Edited* BiallelicEditor embryos Recipients Pregnancies Piglets born (% of born) (% ofborn) 20 ng/μl 60 2 0 NA NA NA TALEN 10 ng/μl 67 2 1  7 0 (0%) 0 (0%)TALEN 2 ng/μl 266 7 5 39 8 (21%) 4 (10%)** TALEN 10 ng/μl 29 1 0 NA NANA ZFN 2 ng/μl 80 2 2  9 1 (11%) 1 (11%)*** ZFN Total 502 14 8 55 9(16%) 5 (9%) *Edited confirmed by sequencing PCR product **Of the 4biallelic TALEN mediated editing events, only 1 was as homozygous event.***The 1 biallelic ZFN mediated event was homozygous. ND—not determinedNA—not appropriate

1. A genetically edited pig comprising a genetic modification to thesequence of the RELA gene which alters the expression or activity of theRELA protein, wherein the modification results in the following changesin the amino acids of the RELA protein: T448A, S485P and S531P. 2-3.(canceled)
 4. The pig of claim 1 in which all cells of the pig containthe genetic modification. 5-10. (canceled)
 11. The pig of claim 1 inwhich the modification is bi-allelic. 12-18. (canceled)
 19. The pig ofclaim 1 wherein the RELA gene has been edited such that it comprises asequence which encodes a RELA protein with a sequence as follows:(SEQ ID NO 9) LLQLQFDADEDLGALLGNNTDPTVFTDLASVDNSEFQQLLNQGVPMPPHTAEPMLMEYPEAITRLVTGSQRPPDPAPTPLGASGLTNGLLPDGEDFSSIA DM.


20. The pig of claim 1 wherein at least a portion of the autologous RELAsequence has been replaced with a sequence which encodes thecorresponding warthog (Phacochoerus sp.) RELA protein sequence.
 21. Thepig of claim 1 in which a portion of the autologous RELA gene has beenremoved and the following corresponding sequence, or a variant thereofwhich encodes the same polypeptide, has been inserted: (SEQ ID NO 11)GTTTGATgCTGATGAGGACCTGGGGGCCCTGCTCGGCAATAACACTGACCCGACCGTGTTCACGGACCTGGCATCCGTCGACAACTCTGAGTTTCAGCAGCTGCTGAACCAGGGTGTAcCcATGCCtCCtCACACAGCcGAGCCCATGCTGATGGAaTAtCCTGAGGCcATAACcCGCTTGGTcACAGGcTCgCAGAGACCtCCCtGCTCCtACTCCtCTGGGGGCCTCgGGGCTgACCAAtGGTcGACC CTCCTCcCcGGGGACGA gGACTTC


22. The pig of claim 1 which demonstrates one or more of the followingphenotypes: an altered NFkappaB signalling capacity; an altered diseaseresilience or tolerance; an altered immune response; and an alteredstress response.
 23. The pig of claim 1 which demonstrates improvedtolerance to one or more of: virus infection; pathogen infection, otherthan viral infection; and general or specific stressors.
 24. The pig ofclaim 1 which has improved tolerance to ASFV infection. 25-45.(canceled)